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Positive and negative populations of cells were determined using unreactive isotype-matched mAbs (Beckman Coulter, Brea, CA) as controls for background staining. Tissues were harvested 4 or 8 hours after IC challenge using a disposable, sterile, 6-mm punch biopsy specimen (Maruho) and cut into halves. All skin samples were snap frozen in liquid nitrogen and stored at ?80��C before use. Total RNA was isolated from frozen Hydroxychloroquine nmr tissue with an RNeasy Fibrous Tissue Mini Kit (Qiagen, Velno, The Netherlands) and then was reverse transcribed into cDNA according to the TaqMan Gene Expression Protocol (Applied Biosystems, Foster City, CA). The expressions of IL-6, TNF-��, and CX3CL1 mRNA were analyzed using a real-time PCR quantification method, according to the manufacturer��s instructions (Applied Biosystems). Sequence-specific primers and probes were designed by Pre-Developed TaqMan assay reagents (Applied Biosystems). The sense primer for mouse IL-6 was 5��-GATGGATGCTACCAAACTGGAT-3��, and the antisense primer was 5��-CCAGGTAGCTATGGTACTCCAGA-3�� (Sigma-Aldrich). The sense primer for mouse TNF-�� was 5��-CCACCACGCTCTTCTGTCTAC-3��, and the antisense primer was 5��-AGGGTCTGGGCCATAGAACT-3�� (Sigma-Aldrich). The sense primer for mouse CX3CL1 was Bioactive Compound Library supplier 5��-ATGACCTCACGAATCCCAGTG-3��, and the antisense primer was 5��-CCGCCTCAAAACTTCCAATGC-3�� (Operon, Alameda, CA). Real-time PCR (one cycle at 50��C for 2 minutes, one cycle at 95��C for 10 minutes, and 40 cycles at 92��C for 15 seconds and at 60��C for 60 seconds) was performed on an ABI Prism 7000 sequence detector (Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize the mRNA. To compare target gene with housekeeping GAPDH gene mRNA expression, the relative expression of real-time PCR products was determined using the ����CT method. One of the control samples was chosen as a calibrator sample. Each sample was examined in duplicate, and the mean CT was used in the equation. Levels of IL-6 and TNF-�� in Chloramben the peritoneal lavage were determined by enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems). Furthermore, serum CX3CL1 levels in patients with PN and healthy controls were measured by a human CX3CL1 ELISA kit (R&D Systems). Ten patients with PN (means �� SD age, 49.5 �� 12.5 years) and 16 healthy control subjects (means �� SD age, 44 �� 16.8 years) were enrolled in this study. The medical ethical committee of the University of Tokyo approved all described studies, and the study was conducted according to Declaration of Helsinki principles. Informed consent was obtained from participants. The U-test was used for determining the level of significance of differences in sample means, and Bonferroni��s test was used for multiple comparisons. P